SUMMARY (PROJECT 3) Recent therapeutic advances have led to incremental improvement in response rates and survival in multiple myeloma. Unfortunately, the disease remains mostly incurable. Anti-CD19 chimeric antigen receptor redirected T cells have been particularly successful in other B-cell malignancies; CD19 is rarely expressed on the malignant plasma cell, though CD19 may be present on rare putative myeloma precursor cells. The B Cell Maturation Antigen, BCMA, is commonly present on neoplastic plasma cells but may not be on the earliest precursors or resistant clones. In patients with advanced multiple myeloma we have observed potent anti- myeloma responses with CAR T cells directed against BCMA (BCMA-CAR) and prolonged responses after CTL019. The depth and durability of these responses are promising, but have varied, and not all patients have had clinical benefit. Treatment failures have been observed in CD19 expressing malignancies such as ALL, CLL and NHL and a number of resistance mechanisms have been observed. In this Project we seek to determine the particular resistance mechanisms that limit CAR T cell therapy for myeloma and develop strategies to overcome them. This will be accomplished in three specific aims. In Aim 1 the frequency and functional clonogenicity of clonotypic B cells will be determined from samples derived from patients treated with CTL019 and BCMA-CAR and correlated with clinical outcome. In Aim 2, we seek to target both early precursors and more differentiated malignant cells to completely eliminate the malignant clone. The feasibility and safety of combining CTL019 and BCMA-CAR will be tested in a clinical trial of patients with advanced myeloma. The degree of depletion of malignant clones and comparison of these clinical results to monotherapy will be assessed. The combined results of Aim 1 and 2 will guide future trials of dual-CAR therapy. In Aim 3 we seek to invent a better anti-myeloma CAR T cell. Detailed immunophenotyping and functional assays of CAR T cells and the tumor environment from samples derived from patients treated on the CAR T trials will be made. These studies will guide the construction of new BCMA-CARs that utilize CRISPR gene-editing to knock-out the most relevant immune checkpoint molecules. This research will be significant because it is aimed at eliminating the myeloma cell of origin and be potentially curative. The innovation of the proposed research lies not just in targeting an antigen commonly on the tumor cell (BCMA) but in addition targeting a second antigen (CD19) on the malignant precursors. The knowledge gained by this project will advance the cellular immunotherapy field.